Menne S, Walz M, Kück U
Lehrstuhl für Allgemeine Botanik, Ruhr-Universität Bochum, Germany.
Appl Microbiol Biotechnol. 1994 Oct;42(1):57-66. doi: 10.1007/BF00170225.
Two cephalosporin genes from Acremonium chrysogenum, pcbAB and pcbC encode the ACV (alpha-aminoadipyl-cysteinyl-valine) synthetase and isopenicillin N-synthetase, respectively. The two adjacent genes are orientated in opposite directions on the chromosomal DNA, separated by a 1.2-kb non-translated sequence, carrying the putative promoter sequences. Complete sequencing of this intergenic region revealed differences from homologous sequences from other strains. To assess the putative promoter strength, we constructed an expression vector carrying the beta-glucuronidase (gusA) and beta-galactosidase (lacZ) genes in opposite orientation. Fusion of the pcbAB-pcbC promoter region resulted in recombinant vector molecules, which were used for in-vivo expression studies. Using the co-transformation procedure, the reporter gene fusions were transferred into A. chrysogenum recipient strains together with vector pMW1. Individual transformants were used for protein preparations to measure specific activities of the enzymes coded by the reporter genes. The data provide in-vivo evidence that the pcbC promoter is at least five times stronger than the pcbAB promoter. Our approach should prove useful in evaluating regulatory sequences that govern gene expression in A. chrysogenum.
来自产黄顶头孢霉的两个头孢菌素基因，pcbAB和pcbC分别编码ACV（α-氨基己二酰-半胱氨酰-缬氨酸）合成酶和异青霉素N合成酶。这两个相邻基因在染色体DNA上呈相反方向排列，被一个携带推定启动子序列的1.2 kb非翻译序列隔开。对该基因间区域进行完整测序发现其与其他菌株的同源序列存在差异。为评估推定启动子的强度，我们构建了一个携带β-葡萄糖醛酸酶（gusA）和β-半乳糖苷酶（lacZ）基因且方向相反的表达载体。pcbAB - pcbC启动子区域的融合产生了重组载体分子，这些分子用于体内表达研究。利用共转化程序，将报告基因融合体与载体pMW1一起转入产黄顶头孢霉受体菌株。单个转化体用于蛋白质制备，以测量报告基因编码的酶的比活性。数据提供了体内证据，表明pcbC启动子比pcbAB启动子至少强五倍。我们的方法在评估产黄顶头孢霉中调控基因表达的调控序列方面应会证明是有用的。
